Isoform specific effects of the autoinhibitory element and the C-terminus of nitr

描述（申请人提供）：一氧化氮合酶在多种生理过程中发挥多种作用，如控制血管张力，中枢神经系统信号转导和免疫反应。时间和/或空间上不适当的一氧化氮（NO）的产生导致几种不同的病理。NOS的内皮异构体（eNOS）控制血管的收缩和扩张，并对血小板聚集有影响，其病理包括高血压和其他血管疾病。自身免疫机制通过iNOS触发NO生成，导致胰岛素依赖型和非胰岛素依赖型糖尿病患者胰腺β细胞凋亡。NOS是一种大型模块化酶，具有含血红素的加氧酶结构域和三结构域还原酶成分。eNOS和nNOS的主要控制是通过调节NADPH到加氧酶活性位点的电子通量来实现的。几种输入中最重要的是钙/钙调素（Ca+2/CaM），但NO的合成也受到磷酸化和蛋白-蛋白相互作用的影响。Ca+2/CaM控制需要位于还原酶区域的控制元件的参与。其中最重要的是FMN结合域的自抑制插入，但C端延伸也有影响。提出的工作是围绕五个假设组织：1。组成型表达NOS （cNOS）的自抑制因子在缺少c端尾部时具有同型特异性作用2。在没有AI的情况下，c端尾部具有同种异构体特异性作用。3. 在ai4存在的情况下，c端尾部具有同种异构体特异性作用。自抑制元件和c端尾相互调节作用。自抑制元件成分具有同种异构体特异性作用。该项目将通过创造新的嵌合基因来检验这些假设，其中控制元件将与远亲NOS酶中的同源物交换。相关的嵌合体已经在多个实验室生产出来，证实了该方法的可行性。通过使用更大的控制序列自然发生的可变性，我们希望大大扩展我们对控制机制的了解。适合本科生参与的实验将用于评估突变体中NO合成和电子转移的控制。更复杂的动力学分析将使我们获得比以前控制元素嵌合体实验更深入的见解。

DESCRIPTION (provided by applicant): Nitric oxide synthases play multiple roles in such diverse physiological processes as control of vascular tone, signal transduction in the central nervous system, and immune response. Temporally and/or spatially inappropriate production of nitric oxide (NO) leads to several different pathologies. The endothelial isoform of NOS (eNOS) controls vascular constriction and dilation and has effects on platelet aggregation, with pathologies including hypertension and other vascular diseases. Autoimmune mechanisms trigger NO production by iNOS, leading to apoptosis of pancreatic beta cells in both insulin-dependent and non-insulin-dependent diabetes mellitus. NOS is a large modular enzyme with a heme containing oxygenase domain and a three domain reductase component. Primary control of eNOS and nNOS is exerted through regulation of electron flux from NADPH to the oxygenase active site. The most important of several inputs is calcium/calmodulin (Ca+2/CaM), but NO synthesis is also influenced by phosphorylation and protein-protein interactions. Ca+2/CaM control requires the participation of control elements located in the reductase region. The most important of these is the autoinhibitory insertion in the FMN binding domain, but the C terminal extension is also influential. The proposed work is organized around five hypotheses: 1. The autoinhibitory element of constitutively expressed NOS (cNOS) has isoform-specific effects in the absence of the C-terminal tail 2. The C-terminal tail has isoform-specific effects in the absence of the AI. 3. The C-terminal tail has isoform-specific effects in the presence of the AI 4. The autoinhibitory element and the C-terminal tail modulate each other's effects 5. Autoinhibitory element components have isoform-specific effect(s). The project will examine these hypotheses by creating novel chimeral genes in which control elements will be exchanged with cognates in distantly related NOS enzymes. Related chimera have been produced in a number of laboratories, establishing the feasibility of the approach. By using a larger section of the naturally occurring variability of the control sequences, we hope to greatly extend our knowledge of the control mechanism. Experiments suitable for undergraduate participation will be used to evaluate control of NO synthesis and electron transfer in mutants. More sophisticated kinetic analysis will allow us to obtain deeper insights than previous experiments with control element chimera.